THE  LIBRARY 

OF 

THE  UNIVERSITY 
OF  CALIFORNIA 

LOS  ANGELES 

GIFT  OF 


Mrs.  Herbert  H.  Huntington 


MEDICAL  WAR   MANUAL  No.  6 

Authorized  by  the  Secretary  of  War 

and  under  the  Supervision  of  the  Surgeon-General 

and  the  Council  of  National  Defense 


Laboratory  Methods 

OF   THE 

UNITED   STATES   ARMY 


COMPILED    BY  THE 

DIVISION     OF     INFECTIOUS    DISEASES 
AND   LABORATORIES 

OFFICE    OF    THE    SURGEON-GENERAL,   WAR    DEPARTMENT 
WASHINGTON,    D.  C. 

SECOND  EDITION,  THOROUGHLY  REVISED 

Illustrates 


LEA  &  FEBIGER 

PHILADELPHIA  AND   NEW  YORK 
1919 


Biomedicd 
Library 


COPYRIGHT 

LEA  &  FEBIGER 

1919 


FOREWORD. 

,WxA/iW^ 

S] 

THIS  edition  of  the  Manual  of  Laboratory  Methods 
represents  a  complete  revision  of  the  first  edition.  The 
chapter  on  special  bacteriological  methods  has  been  almost 
entirely  revised,  with  the  addition  of  certain  methods  wrhich 
have  proved  practicable  in  the  Army  laboratories.  The 
chapter  on  sanitary  examination  of  water  and  sewage  has 
been  largely  revised.  A  complete  new  section  on  autopsy 
technique,  with  directions  for  preparation  and  shipment  of 
museum  specimens,  has  been  added.  Many  minor  sections 
have  been  introduced,  taking  advantage  of  the  experience 
gained  in  the  laboratories  both  in  this  country  and  in  France. 
Where  a  method  adopted  as  a  standard  by  the  Central  Labo- 
ratory of  the  A.  E.  F.  has  differed  from  that  used  in  the  Army 
in  this  country,  both  methods  are  given.  Special  acknowl- 
edgment is  made  to  Dr.  Donald  D.  Van  Slyke,  Dr.  William 
G.  MacCallum,  Captain  Oswald  T.  Avery  and  Major  Edward 
T.  Tucker,  for  aid  hi  the  preparation  of  some  of  the  larger 
sections. 

THE  EDITOR. 


6*76739 


(v) 


CONTENTS. 


INTRODUCTION 

GENERAL  RULES  FOR  THE  CONDUCT  OF  THE  LABORATORY  . 
COLLECTION  AND  SHIPMENT  OF  SPECIMENS  AND  MATERIALS 
SOLUTIONS  AND  STAINS 

CLINICAL  PATHOLOGICAL  WORK 

Routine  Methods 

Preparation  of  Salvarsanized  Serum 

Method  of  Determining  Blood  Groups 

Preparation  of  Microscopic  Specimens 

Technique  for  Wassermann  Test 

GENERAL  AUTOPSY  METHODS 

GENERAL  BACTERIOLOGICAL  METHODS 

SPECIAL  BACTERIOLOGICAL  METHODS 

Determination  of  Types  of  Pneumococcus 

Method  for    Isolation    and    Identification    of   Streptococcus 

Hemolyticus 

Standard  Technique  of  Meningococcus  Carriers       .... 

Bacillus  of  Diphtheria 

Bacillus  of  Tetanus 

Tubercle  Bacillus 

Bacillus  of  Anthrax 

Gas  Gangrene  and  Anaerobic  Bacillus  in  Wounds    .... 
Bacillus  of  the  Typhoid,  Paratyphoid  and  Dysencery  Group  . 

Cholera 

Bacillus  of  Bubonic  Plague 

Bacillus  of  Glanders 

Bacillus  of  Influenza 

Rabies 

Detection  of  the  Spirocheta  Icterohemorrhagiae        .... 

Bacteriology  of  Ropy  Bread 

(vii) 


10     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

THE   RESPONSIBILITIES   OF  THE   LABORATORY. 

The  functions  of  the  laboratory  consist  chiefly  in  helping 
to  safeguard  the  health  of  the  troops  by  making  rapid  and 
accurate  diagnosis  of  infectious  and  other  diseases,  by  which 
the  division  surgeon  and  his  staff  may  be  guided  in  prophyl- 
axis and  treatment. 

The  laboratory  director  should  bear  in  mind  that  military 
laboratories  must  combine  the  functions  of  Health  Depart- 
ment laboratories  with  those  of  diagnostic  hospital  labora- 
tories. 

The  directors  of  such  laboratories  are  responsible  directly 
to  the  commanding  officer  of  the  camp  base  hospital, 
through  whom  he  will  cooperate  with  the  sanitary  inspector 
of  the  camp  in  any  sanitary  examinations  in  which  the  labora- 
tory can  facilitate  control  of  camp  conditions. 

The  director  of  the  laboratory  is  responsible  not  only  for 
the  proper  conduct  of  the  laboratory  and  the  accuracy  of 
the  reports  which  are  issued,  but  also  for  the  preparation 
of  requisitions  for  the  supplies  which  are  used  in  the  labora- 
tory. 

Such  requisitions  are  made  up  in  accordance  with  the 
Manual  of  the  Medical  Department,  paragraph  477  to  490,  and 
he  should  note  especially  paragraph  485. 


GENERAL  RULES  FOR  THE  CONDUCT  OF  THE 
LABORATORY. 

1.  Wear  apron  or  other  protective  covering  over  uniform 
when  in  the  laboratory.     This  avoids  carrying  infectious 
material  to  mess. 

2.  Keep  your  desk  and  floor  area  clean. 

3.  Wash  and   disinfect  your  hands  before  leaving  the 
laboratory. 


INTRODUCTION  11 

4.  Preserve  all  cultures,  except  liquefied  gelatin  plates,  for 
one  week  before  discarding. 

5.  Place  all  discarded  cultures  and  slides  in  receptacles 
provided  for  that  purpose.    If  they  are  not  provided,  obtain 
them. 

6.  Label  everything  and  do  it  legibly. 

7.  If  a  culture  be  dropped  on  the  floor,  breaking  the  glass, 
do  not  clean  it  up  until  everything  has  been  disinfected  for 
one  hour;  wet  a  towel  in  antiseptic  solution  and  cover  the 
entire  infected  area. 

8.  Clean  the  oil-immersion  lens  before  leaving  the  labora- 
tory. 

9.  Keep  all  room  temperature  cultures  in  dark,  dust-free 
closets. 

10.  Do  not  keep  gas  burners  lighted  when  not  in  use.    Do 
not  allow  sterilizers  and  water  baths  to  boil  dry.     Do  not 
leave  stoppers  out  of  bottles.     Do  not  take  more  media, 
stains,  and  other  supplies  than  you  need. 

11.  Clean  oil  from  the  condenser,  lens,  and  mirror  of  the 
dark-field  apparatus  before  leaving  for  the  day.    If  you  do 
not,  it  will  have  to  be  done  in  the  morning  before  the 
apparatus  can  be  used,  and  will  then  be  much  more  difficult. 

12.  Remember  that  it  is  as  important  to  keep  accurate 
records  as  to  carry  on  accurate  tests. 

The  following  tabulation  is  suggested  as  a  general  plan  for 
the  organization  of  the  laboratory: 

1.  The  Chief  of  the  Laboratory  should  organize  his  depart- 
ment so  that  he  will  be  relieved  of  routine  work,  will  have 
time  to  act  as  consultant  on  the  laboratory  findings  to  the 
hospital  and  to  the  Camp  Surgeon,  as  well  as  have  time  for 
administrative  detail. 

2.  It  is  suggested  that  the  laboratory  personnel  be  divided 
into  groups  with  an  individual  with  special  training  along  a 
specific  line  in  charge  of  each  group.     For  types  of   work 
requiring  technical  skill  and  experience,  such  as  pneumo- 


12     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

coccus  grouping,  it  is  considered  advisable  to  have  one  person 
responsible  and  held  to  this,  so  as  to  obtain  uniformity  of 
results. 

3.  The  following  plan  for  division  of  work  is  suggested: 
The  personnel  should  be  divided  as  far  as  possible  according 
to  training  and  the  amount  of  work  to  be  accomplished  in  the 
different  groups. 

(a)  Pathology: 

Autopsy. 

Section  cutting  (special  technician). 

Preservation  and  shipment  of  museum  specimens. 

(b)  Clinical  microscopy: 

Routine  examination  of  blood,  sputum,  urine,  feces, 
spinal  fluid  (cell  counts),  urethral  discharges, 
dark  field  examinations,  etc. 

(c)  Bacteriology  and  serology: 

Preparation  of  media. 

Bacteriology  of  sputum,  pneumonia  and  strepto- 
coccus typing  (special  technician). 

Nasopharyngeal  cultures  and  detection  of  carriers 
of  meningococcus  and  diphtheria. 

Bacteriology  of  feces  and  urine  for  detection  of 
typhoid  and  dysentery  groups. 

Bacteriology  of  food,  milk,  water  and  sewage. 

Blood  cultures. 

Wassermann  tests. 

(d)  Chemistry: 

Clinical  chemistry,  special  examanition  of  blood, 

urine,  etc. 
Chemical  examination  of  food,   milk,   water  and 

sewage. 

(e)  Records  and  reports: 

Files  and  librarian. 
Journals  and  books. 
(/)   Photographer: 

For  cases,  specimens,  gross  and  microscopic. 


COLLECTION  AND  SHIPMENT  OF 
SPECIMENS  AND  MATERIALS. 


SPUTUM. 

THE  sputum  from  the  lower  respiratory  passages,  and  not 
saliva  or  the  nasopharyngeal  secretions,  should  be  sub- 
mitted. No  disinfectant  should  be  added  to  the  specimen. 
It  is  necessary  to  explain  this  to  the  ward  attendants,  as  they 
are  usually  instructed  to  add  disinfectants  to  all  discharges. 
Gross  contamination  should  be  avoided  by  keeping  the  speci- 
men bottle  tightly  corked  except  during  actual  collection 
of  the  specimen.  Plainly  label  the  bottle  with  the  patient's 
name,  rank,  organization,  the  station  from  which  sent,  and 
the  examination  desired.  Each  specimen  must  be  accom- 
panied by  requests,  in  duplicate,  on  Form  550,  M.  D. 

FECES. 

The  specimen  bottles  mentioned  in  this  circular  are  for 
forwarding  specimens  of  feces  for  examination  for  parasites 
and  their  ova  and  occult  blood.  If  bacteriological  examina- 
tions for  typhoid  fever,  paratyphoid  fever,  or  bacillary 
dysentery  are  desired,  specimens  should  be  forwarded  in  glass- 
stoppered  bottles.  Patients  in  whose  cases  an  examination 
for  occult  blood  is  desired  should  be  placed  on  a  meat-free 
diet  for  at  least  two  days  prior  to  the  collection  of  the  specimen. 

The  specimens  or  feces  are  to  be  collected  in  large  num- 
bers for  carrier  examinations  for  typhoid,  paratyphoid,  etc. 
Time  can  be  economized  and  a  system  for  laboratory  col- 
lections established  by  furnishing  test-tubes  containing 

(13) 


14     LA  BORA  TOR  Y  ME  THODS  OF  UNI  TED  S  TA  TES  ARMY 

sterile  swab  sticks.  The  specimen  feces  can  be  taken  up 
with  a  swab  stick  and  inserted  into  the  test-tubes,  upon  which 
are  labels  to  be  used  for  the  name,  rank  and  organization 
of  the  subject. 

URINE. 

Specimens  of  urine  for  bacteriological  examination  or 
animal  inoculation  must  be  collected  under  aseptic  condi- 
tions. Catheterization  should  be  resorted  to  and  no  disin- 
fectant should  be  added.  Specimens  to  be  examined  for 
organisms  of  the  typhoid-paratyphoid  group  should  be 
forwarded  in  bile  medium.  Each  specimen  of  urine  (except 
for  typhoid)  must  be  accompanied  by  requests,  in  duplicate, 
on  Form  55m,  M.  D. 

USE  OF  DIPHTHERIA  CULTURE  TUBES. 

Good  illumination  of  the  throat  is  essential.  Remove  the 
swab  from  its  container,  and  while  depressing  the  tongue, 
pass  swab  into  pharynx,  avoiding  contact  with  tongue  or 
other  parts  of  mouth,  and  rub  firmly,  but  gently,  over  any 
visible  membrane,  or  if  no  membrane  is  present,  over  the 
tonsils  and  pharynx.  Withdraw  swab  and  rub  it  over  the 
whole  surface  of  the  Loeffler  serum  medium,  being  careful 
that  the  portion  of  the  swab  previously  in  contact  with  the 
membrane  comes  in  contact  with  the  medium.  The  inocula- 
tion of  the  medium  should  be  thorough,  but  the  surface  of  the 
medium  must  not  be  broken. 

Replug  the  culture  tube,  return  the  swab  to  its  container, 
pack  securely  in  cotton,  and  mail  to  the  officer  in  charge  of 
the  laboratory. 

The  culture  tube  must  be  plainly  labelled  with  the  name, 
rank  and  organization  of  the  patient  and  the  station  from 
which  the  culture  is  sent.  The  nature  of  the  examination 
desired — "for  diphtheria" — must  also  be  shown.  Requests, 
in  duplicate,  on  Form  55u  must  accompany  each  culture. 


COLLECTION  OF  SPECIMENS  AND  MATERIALS          15 

COLLECTING  AND  SHIPPING  SAMPLES  OF  MILK  FOR 
CHEMICAL  AND  BACTERIOLOGICAL  EXAMINATION. 

COLLECTION  OF  SAMPLES. — The  surgeon  should  request, 
through  the  commanding  officer,  that  the  delivery  wagon  of 
each  dairyman  supplying  the  command  be  directed  to  report 
at  the  surgeon's  office  at  a  designated  day  each  month  for 
the  purpose  of  delivering  milk  samples. 

A  one-quart  bottle  should  be  selected  at  random  for 
analysis,  the  bottle  being  labelled  with  the  name  of  the  dairy. 

PREPARATION  FOR  SHIPMENT. — If  samples  are  delivered 
during  the  early  morning  hours  they  should  be  placed  on 
ice  immediately.  All  samples  should  be  forwarded  to  the 
laboratory  on  the  day  of  collection. 

The  following  procedure  should  be  carried  out  in  preparing 
the  samples  for  shipment:  The  quart  of  milk  must  be  poured 
25  times  between  the  original  container  and  a  sterile  bottle 
or  flask  in  order  that  the  milk  and  cream  may  be  thoroughly 
mixed  and  that  clumps  of  bacteria  may  be  broken  up.  After 
thorough  mixing  add  i  c.c.  of  commercial  formalin  to  the 
quart  (1000  c.c.)  of  milk  and  agitate  thoroughly  to  ensure 
inhibition  of  further  growth  of  bacteria.  Then  fill  the  sterile 
60  c.c.  sample  bottle.  When  it  is  desired  to  learn  the  type 
of  organisms  present  the  formalin  should  be  omitted. 

It  is  essential  that  the  bottle  containing  the  sample  be 
filled  flush  to  the  lower  end  of  the  stopper  to  prevent  churning 
of  sample  with  formation  of  butter  while  in  transit  to  the 
laboratory.  Seal  with  paraffin  or  wax  and  cover  with  a 
square  of  muslin  held  in  place  by  copper  wire. 

Label  each  bottle  with  the  name  of  station  or  command, 
location  and  name  of  dairy,  date  of  collection  and  date  of 
shipment.  Pack  securely  in  absorbent  cotton  to  avoid 
breakage. 

Samples  of  milk  for  examination  must  reach  the  laboratory 
prior  to  the  25th  of  each  month.  Milk  samples  other  than 
routine  may  be  sent  to  the  laboratory  when  occasion  demands. 


18     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

labelled  with  the  patient's  name,  rank,  organization,  and  the 
station  from  which  the  specimen  is  sent.  The  test  desired 
(Wassermann,  gonococcus-fixation,  etc.)  should  be  desig- 
nated also.  The  capsules  should  be  wrapped  securely  in 
cotton  to  avoid  breakage  in  the  mails. 

(g)  Specimens  for  the  Wassermann  test  must  be  accom- 
panied by  requests,  in  duplicate,  on  Form  55q,  M.D.,  and  the 
first  time  the  serum  of  an  individual  is  tested  at  this  laboratory 
it  must  also  be  accompanied  by  a  Wassermann  card,  Form 
97,  M.  D. 

AGGLUTINATION  TESTS. — The  institution  of  prophylactic 
inoculation  against  typhoid  and  paratyphoid  fevers  has 
very  largely  obviated  the  usefulness  of  the  agglutination 
test  (Widal)  as  a  diagnostic  procedure  in  these  diseases.  The 
results  are  of  value  in  establishing  a  positive  diagnosis  in 
inoculated  individuals  only  when  there  is  a  definite  increase 
in  the  agglutinating  property  of  the  serum,  as  shown  by 
repetition  of  the  test  with  sera  collected  at  intervals  of  a 
week  or  ten  days.  For  early  diagnosis  the  blood  culture  should 
be  resorted  to  in  all  cases.  In  bacillary  dysentery  the  agglu- 
tination test  may  be  of  value  in  establishing  a  diagnosis; 
but,  as  a  rule,  the  serum  possesses  agglutinating  properties 
only  in  the  severe  or  moderately  severe  cases.  Isolation  of 
the  causative  organism  by  bacteriological  examination  of 
the  feces  is  a  surer  and  altogether  more  satisfactory  diag- 
nostic procedure. 

Blood  specimens  forwarded  for  agglutination  tests  for 
typhoid  and  paratyphoid  fevers  should  be  collected  in 
Wright's  capsules,  under  aseptic  precautions,  in  order  that 
cultures  may  be  attempted  from  the  clot. 

COLLECTING   AND   FORWARDING   SPECIMENS   FOR   THE 
DIAGNOSIS   OF   GLANDERS. 

CULTURES. — The  glanders  bacillus  can  be  obtained  easily, 
in  pure  cultures,  from  the  interior  of  suppurating  glands  and 


COLLECTION  OF  SPECIMENS  AND  MATERIALS          19 

nodules,  which  have  not  yet  opened  to  the  surface.  The  dis- 
charges from  the  nostrils,  or  from  an  open  lesion,  are  much 
less  satisfactory,  as  very  few  bacilli  may  be  present,  and  the 
detection  of  these  is  difficult  because  of  the  invariable 
admixture  of  numerous  other  microorganisms.  Glycerin-agar 
slants  should  be  used.  The  procedure  of  making  cultures 
follows: 

(a)  Select  a  fluctuating  gland  or  nodule  which  has  not  yet 
opened  to  the  surface,  shave  the  overlying  skin,  and  sterilize 
this  area  with  a  thick  coating  of  tincture  of  iodin. 

(6)  Incise  the  nodule  with  a  sterile  scalpel. 

(c)  Evert  the  edges  of  the  incision  with  thumb  and  middle 
finger,  introduce  a  sterile  swab  into  the  center  of  the  lesion, 
and,  exercising  care  that  it  does  not  touch  the  skin  edge  of 
the  wound,  rotate  it  gently  so  as  to  thoroughly  impregnate 
it  with  the  contents  of  the  lesion. 

(d)  Remove  the  cotton  plug  from  the  culture  tube,  flame 
the  neck  of  the  tube  and  smear  the  material  on  the  swab  over 
the  surface  of  the  culture  medium. 

(e)  Carefully  replace  the  cotton  plug  in  the  culture  tube, 
return  the  swab  to  the  tube  in  which  it  was  received  and 
forward  both  tubes  to  the  laboratory. 

(/)  The  presence  of  B.  mallei  in  material  from  open  lesions, 
nasal  discharge  and  sputum  (pulmonary  lesions  in  man)  can 
be  determined  by  guinea-pig  inoculation  (Straus  reaction). 
Collect  discharge  and  forward  in  sealed  container;  if  neces- 
sary, add  a  small  amount  of  saline  to  prevent  drying. 

(g)  The  tubes  or  containers  should  be  plainly  labelled  with 
name  (human  case)  or  name  or  identification  mark  of  animal, 
the  nature  of  the  material,  the  examination  requested,  to- 
gether with  the  name,  rank  and  station  of  the  veterinarian 
to  whom  the  reports  of  the  examination  are  to  be  forwarded. 
Requests,  in  duplicate,  on  Form  55U,  M.  D.,  must  accompany 
each  culture. 

COMPLEMENT-FIXATION  OR  AGGLUTINATION  TEST. — A  posi- 

2 


20     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

tive  complement-fixation  test  may  be  obtained  from  the 
seventh  to  the  tenth  day  and  usually  persists  during  the 
course  of  the  disease.  The  agglutination  test  may  be  positive 
in  four  to  seven  days,  the  content  in  agglutinins  increasing 
early  in  the  disease,  but  decreasing  if  the  disease  becomes 
chronic.  All  blood-serum  tests  are  influenced  by  the  injec- 
tion of  mallein  or  vaccine,  and  blood  should  be  taken  before 
their  administration. 

|t  Blood  for  these  tests  is  collected  from  man  as  per  instruc- 
tions under  Wassermann,  page  16;  from  horse  as  follows: 

(a)  Sterilize  a  large-sized  hypodermic  needle  by  boiling. 
Do  not  use  phenol  or  other  antiseptics  for  this  purpose. 

(b)  Shave  and  sterilize  with  tincture  of  iodin  the  skin  over 
the  jugular  vein. 

(c)  Make  the  vein  prominent  by  pressing  with  the  thumb 
below  the  area  selected  for  puncture,  thrust  needle  into  vein 
and  permit  blood  to  flow  into  sterile  bottle  furnished  for  this 
purpose.    Tightly  cork  after  collection. 

Stand  toward  the  side  of  the  horse,  while  drawing  blood, 
to  avoid  danger  from  sudden  rearing. 

Label  specimen  plainly  with  name  (human  case)  or  name 
or  identification  mark  of  animal,  the  nature  of  the  specimen, 
the  test  desired  and  the  name,  rank  and  station  of  the  surgeon 
or  veterinarian  to  whom  report  is  to  be  returned.  Pack  the 
bottle  securely  in  cotton  to  avoid  breakage.  Requests  in 
duplicate,  on  Form  5$u,  M.  D.,  must  accompany  each  speci- 
men. 

WATER  ANALYSIS,  BACTERIOLOGICAL.    DIRECTIONS  FOR 
COLLECTING  AND  SHIPPING. 

These  directions  are  a  transcription  of  the  instructions  in 
the  Manual  of  the  Medical  Department,  1916: 

356.  At  the  time  of  forwarding  the  water  the  officer  to 
whom  it  is  sent  should  be  advised  of  the  following  particulars: 
(i)  The  date,  place  and  mode  of  shipment;  (2)  the  date 


COLLECTION  OF  SPECIMENS  AND  MATERIALS         21 

and  place  of  the  collection  of  the  water;  (3)  the  character 
of  the  watershed,  its  topography,  and  the  uses  to  which  the 
country  is  put, if  inhabited;  (4)  the  proximity  of  houses, 
barns,  privies,  or  other  possible  sources  of  contamination 
to  the  place  of  collection  or  the  source  of  supply;  (5)  the 
proximity  of  fertilized  land  to  such  place  or  source  and 
whether  the  said  land  is  higher  or  lower  than  the  adjacent 
land;  (6)  such  other  information  as  may  suggest  a  possible 
deleterious  influence  on  the  purity  of  the  water.  If  the  water 
is  from  a  well  the  letter  should  report  the  depth  of  the  wrell, 
the  strata  found  in  digging  or  boring  it,  and  the  depth  of  the 
water  in  the  well. 

357.  The  specimen  should,  when  practicable,  be  collected 
by  a  medical  officer.    If  the  water  to  be  examined  is  delivered 
through  pipes  or  is  pumped  from  a  well  or  cistern  the  local 
supply  pipe  and  all  pump  connections  should  be  emptied 
by  allowing  the  water  to  run  for  fifteen  minutes  before  taking 
the  samples. 

358.  BACTERIOLOGICAL  EXAMINATIONS. — Samples  of  water 
for  bacteriological  examination  should  be  collected  in  bottles 
furnished  for  the  purpose.    Each  bottle  is  sterilized  before 
leaving  the  laboratory,  and  the  glass  stopper  is  protected 
by  a  piece  of  heavy  sterilized  muslin  securely  wired  to  the 
neck  of  the  bottle.    The  stopper  should  not  be  removed  until 
immediately  before  the  bottle  is  filled. 

(a)  In  taking  specimens  from  a  faucet  or  pump  (after 
emptying  the  supply  pipes  and  connections  conformably  to 
paragraph  357)  a  small,  gentle  stream  should  be  allowed 
to  flow,  the  stopper  taken  out,  the  bottle  grasped  near  the 
bottom,  held  in  an  upright  position,  and  the  stream  permitted 
to  flow  into  the  bottle  until  it  is  filled  to  the  shoulder.  The 
stopper  should  then  be  replaced;  both  it  and  the  cloth  should 
be  secured  by  carrying  the  wire  several  times  around  the  neck 
of  the  bottle  and  twisting  the  ends  tight.  The  stopper  must 
be  handled  only  by  the  square  cloth-covered  top.  The  lip 


22     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

of  the  bottle  must  not  be  brought  in  contact  with  the  faucet 
or  spout,  nor  should  the  neck  of  the  bottle  or  naked  part  of 
the  stopper  be  permitted  to  come  in  contact  with  any  object 
during  the  manipulation.  The  projecting  flange  is  designed 
to  protect  the  plug  of  the  stopper,  which  it  will  do  if  the 
stopper,  after  withdrawal,  is  held  by  the  top  in  a  vertical 
position.  The  stopper  should  not  be  laid  down  and  the  cloth 
should  not  be  handled  by  the  fingers  except  in  the  act  of 
securing  the  wire  about  it.  When  well  water  is  to  be  examined 
the  bottle  should  be  filled  directly  from  the  bucket  constantly 
in  use  for  drawing  the  water,  and  from  no  other  vessel. 

(b)  On  account  of  the  labor  involved  and  the  possibility 
of  error,  bacteriological  examinations  of  water  collected  in 
any  other  than  the  prescribed  receptacles  will  not  be  made. 

(c)  Each  package  should  be  plainly  marked  to  show  the 
source  from  which  the  samples  are  taken  and  the  date  of 
collection. 

(d)  The  case  should  be  marked  "Water  for  Bacteriological 
Examination,"  and  it  should  be  forwarded  by  mail  at  the 
earliest  moment. 

(See  paragraph  355a:  All  bottles  containing  fluid  material 
sent  through  the  mails  must  be  securely  packed  in  cotton  in 
double  containers.) 

PREPARATION  AND  SHIPMENT  OF  AUTOPSY  SPECIMENS  FOR 
THE  MUSEUM. 

See  page  109 


SOLUTIONS  AND  STAINS. 


PHYSIOLOGICAL   SALT   SOLUTION. 

FOR  bacteriological  work,  physiological  solution  is  usually 
made  up  by  adding  8.5  grams  of  sodium  chloride  to  a  liter 
of  distilled  water.  When  for  reasons  of  speed  and  convenience 
tap  water  is  used,  one  should  have  some  idea  of  the  salt 
contents  of  the  water  used  before  relying  upon  it. 

SODIUM    CITRATE   SOLUTION. 

For  bacteriological  purposes  this  contains  i  per  cent,  of 
sodium  citrate  and  0.85  per  cent,  of  sodium  chloride. 

If  sodium  citrate  is  to  be  used  for  prevention  of  coagulation 
of  blood  without  considerably  changing  the  volume  of  the 
blood  the  solution  is  made  up  to  contain  10  per  cent,  of 
sodium  citrate  and  0.85  sodium  chloride. 

FIVE   PER    CENT.    SULPHURIC   ACID   FOR   DECOLORIZING 
(AS  USED  IN  TUBERCLE  BACILLUS  STAINING). 

Slowly  allow  2.7  c.c.  of  c.  p.  sulphuric  acid  of  a  specific 
gravity  of  1.84  to  flow  into  80  c.c.  of  distilled  water.  After 
cooling,  bring  volume  up  to  100  c.c.  (St.  Luke's  Manual.) 

ACID   ALCOHOL   (ORTH). 

HC1 I.OC.C. 

70  per  cent,  alcohol 99.0  c.c. 

(23) 


24     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 
OXALATE  SOLUTION  FOR  BLOOD  CULTURE. 

Ammonium  oxalate 2.0  grams 

Sodium  chloride   ......          6.0  grams 

In  distilled  water 1000.0  c.c. 

ZENKER'S  FLUID. 
Potassium  dichromate     ....  2.5  grams 

Mercury  bichloride 5.0  grams 

Water 100.0  c.c. 

Glacial  acetic  acid  5  per  cent,  is  added  to  this  stock  solu- 
tion just  before  use. 

COPPER  SULPHATE  SOLUTION  FOR  CAPSULE  STAIN. 

20  grams  of  copper  sulphate  crystals  are  dissolved  in  100  c.c. 
of  water. 

STOCK  STAINING  SOLUTIONS. 

It  is  convenient  in  stationary  laboratories  to  keep  stock 
stains  in  the  form  of  saturated  solutions.  The  strengths 
of  various  saturated  solutions  are  as  follows: 

Saturation  strengths. 

Fuchsin  in  alcohol 3.0  per  cent. 

Gentian  violet  in  water  .      .      .      .     1.5        " 
Gentian  violet  in  alcohol      .     .      .     4.8 
Methylene  blue  in  water      .      .      .6.7        " 
Methylene  blue  in  alcohol    .      .      .     7.0        " 

Safranin .      .4.0 

Saturated  alcoholic  solutions  can  be  kept  and  aqueous 

staining  solutions  can  best  be  made  by  adding  5  per  cent,  of 

the  filtered  alcoholic  solutions  to  water. 

LOEFFLER'S  ALKALINE  METHYLENE  BLUE. 

Saturated  alcoholic  solution  of  methyl- 

ene  blue 30.0  c.c. 

i  to  10,000  solution  potassium  hydrox- 
ide in  water  i oo. o  c.c. 


SOLUTIONS  AND  STAINS  25 

ZIEHL  CARBOL-FUCHSIN  SOLUTION. 

Fuchsin  (basic) i.ogram 

Alcohol  (absolute) 10 .  o  c.c. 

5  per  cent,  phenol loo.oc.c. 

To  make  up  this  staining  solution  by  another  method, 
90  c.c.  of  a  5  per  cent,  aqueous  solution  of  phenol  is  mixed 
with  10  c.c.  of  saturated  alcoholic  basic  fuchsin. 

CAPSULE  STAINS. — Hiss's  Copper  Sulphate  Method. — Cover- 
slip  preparations  are  made  by  smearing  the  organisms  in  a 
drop  of  animal  serum,  preferably  beef-blood  serum. 

Dry  in  air  and  fix  by  heat. 

Stain  for  a  few  seconds  with — 

Saturated  alcoholic  solution  of  fuchsin  or  gentian  violet, 
5  c.c.,  in  distilled  water,  95  c.c.  This  combination  is  often 
too  weak  for  good  results.  A  gentian-violet  or  fuchsin 
solution  twice  as  strong  is  advantagenous.  Gram's  gentian 
violet  or  carbol-fuchsin  can  be  used. 

The  cover-slip  is  flooded  with  the  dye  and  the  preparation 
held  for  a  second  over  a  free  flame  until  it  steams. 

Wash  off  dye  with  20  per  cent,  aqueous  copper  sulphate 
solution. 

Blot  (do  not  wash). 

Dry  and  mount. 

NEISSER  STAIN  FOR  POLAR  BODIES. 

Methylene  blue i .  o  gram 

Absolute  alcohol 200.0  c.c. 

Glacial  acetic  acid       .      .     .  '  .     .  50 .  o  c.c. 

Distilled  water looo.oc.c. 

Preparations  fixed  by  heat  are  immersed  in  this  stain  for 
five  seconds  and  then  washed  in  water  and  counterstained 
with  Bismarck  brown  or,  better,  safranin. 


26    LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

CARBOL-THIONIN. 

Saturated  solution  of  thionin  in  50  per 

cent,  alcohol lo.oc.c. 

Two  per  cent,  phenol loo.oc.c. 

Stain  for  two  minutes. 

GRAM'S  METHOD  AND  MODIFICATIONS. — Preparations  are 
made  on  cover-slips  or  slides  in  the  usual  way. 

It  is  always  necessary  to  control  Gram  stains  with  organ- 
isms of  known  type. 

The  preparation  is  covered  with  an  anil  in  gentian-violet 
solution,  which  is  best  made  up  freshly  before  use. 

The  staining  fluid  is  made  up,  according  to  Gram's  original 
directions,  as  follows: 

Five  c.c.  of  anilin  oil  are  shaken  up  thoroughly  with  125 
c.c.  of  distilled  water.  This  solution  is  then  filtered  through 
a  moist  filter  paper. 

To  1 08  c.c.  of  this  anilin  water  add  12  c.c.  of  a  saturated 
alcoholic  solution  of  gentian  violet.  The  stain  acts  best 
when  twelve  to  twenty-four  hours  old,  but  may  be  used  at 
once.  It  lasts,  if  well  stoppered,  for  three  to  five  days.  A 
more  convenient  and  simple  method  of  making  up  the  stain 
is  as  follows: 

To  10  c.c.  of  distilled  water  in  a  test-tube  add  anilin  oil 
until  on  shaking  the  emulsion  is  opaque — roughly,  i  to  10. 
Filter  this  through  a  wet  paper  until  the  filtrate  is  clear. 
To  this  add  saturated  alcoholic  solution  of  gentian  violet 
until  the  mixture  is  no  longer  transparent  and  a  metallic 
film  on  the  surface  indicates  saturation.  One  part  of  alco- 
holic saturated  gentian  violet  to  nine  parts  of  the  anilin  water 
will  give  this  result.  This  mixture  may  be  used  immediately 
and  lasts  two  to  five  days  if  kept  in  a  stoppered  bottle. 

Cover  the  preparation  with  this ;  leave  on  for  five  minutes. 
Pour  off  excess  stain  and  cover  with  Gram's  iodin  solution 
for  two  or  three  minutes. 


SOLUTIONS  AND  STAINS 


27 


GRAM'S  IODIN  SOLUTION. 

lodin i .  o  gram 

Potassium  iodide 2.0  grams 

Distilled  water 300 .  o  c.c. 

Decolorize  with  97  per  cent,  alcohol  until  no  further  traces 
of  the  stain  can  be  washed  out  of  the  preparation.  This  takes 
usually  thirty  seconds  to  two  minutes,  according  to  thinness 
of  preparation.  Wash  in  water. 

Counterstain  with  an  aqueous  contrast  stain,  preferably 
Bismarck  brown  or  safranin. 

Sterling's  Modification  of  Gram's  Method. — Two  c.c.  anilin 
oil  and  10  c.c.  95  per  cent,  alcohol.  Shake  and  add  88  c.c. 
distilled  water.  Five  grams  of  gentian  violet  are  ground  in  a 
mortar  and  the  anilin  solution  added  slowly  while  grinding. 
Filter.  This  solution  keeps  and  stains  in  one-half  to  one 
minute. 

CLASSIFICATION  or  THE  MOST  IMPORTANT  PATHOGENIC 
BACTERIA  ACCORDING  TO  GRAM'S  STAIN. 


Gram-positive. 
(Retain  the  gentian  violet.) 
Micrococcus  pyogenes  aureus 
Micrococcus  pyogenes  albus 
Streptococcus  pyogenes 
Micrococcus  tetragenus 
Pneumococcus 
Bacillus  subtilis 
Bacillus  anthracis 
Bacillus  diphtherias 
Bacillus  tetanus 
Bacillus  tuberculosis  and  other 

acid-fast  bacilli 
Bacillus  aerogenes  capsulatus 
Bacillus  botulinus 


Gram-negative. 
(Take  counterstain.) 
Meningococcus 
Gonococcus 

Micrococcus  catarrhalis 
Bacillus  coli 
Bacillus  dysenterise 
Bacillus  typhosus 
Bacillus  paratyphosus 
Bacillus  fecalis  alkaligenes 
Bacillus  enteritidis 
Bacillus  proteus 
Bacillus  mallei 
Bacillus  pyocyaneus 
Bacillus  influenzas 
Bacillus  mucosus  capsulatus 
Bacillus  pestis 
Bacillus  maligni  edematis 
Spirillum  choleras 
Bacillus  Koch-Weeks 
Bacillus  Morax-Axenfeld 


28    LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

CARBOL-GENTIAN  VIOLET. 

Saturated  alcoholic  solution  of  gentian 

violet 90.0  c.c. 

Five  per  cent,  phenol  in  water       .      .     1000 .  o  c.c. 

This  solution  retains  its  staining  powers  for  the  Gram 
method  of  staining  for  a  longer  period  than  does  the  ordinary 
Gram  solution,  but  is  not  as  permanent  as  the  Sterling 
modification. 

POLYCHROME  STAINS. — The  Romanowsky  stain  depends 
on  the  formation  of  methylene  azure  and  methylene  violet 
in  alkaline  solution  of  methylene  blue.  When  this  solution 
is  mixed  with  a  solution  of  water-soluble  yellowish  eosin, 
the  eosinates  of  methylene  azure,  methylene  violet,  and 
methylene  blue  are  thrown  down,  as  these  eosinates  are 
insoluble  in  water. 

Wright's  stain  consists  of  a  solution  of  these  eosinates  in 
methyl  alcohol. 

Any  methylene  blue  and  any  yellowish  water-soluble  eosin 
issued  by  the  Medical  Department  can  be  used  in  preparing 
the  stain. 

WRIGHT'S  STAIN. — Add  i  gram  of  methylene  blue  to  100 
c.c.  of  a  0.5  per  cent,  solution  of  sodium  bicarbonate  in  water 
and  heat  for  one  hour,  after  steam  is  up,  in  an  Arnold  sterilizer. 
The  flask  containing  the  alkaline  methylene  blue  solution 
should  be  of  such  size  that  the  depth  of  the  fluid  does  not 
exceed  two  and  a  half  inches.  When  cool  add  to  the  methyl- 
ene blue  solution  500  c.c.  of  a  i  to  1000  eosin  solution  (yel- 
lowish eosin,  water  soluble).  Add  the  eosin  solution  slowly, 
stirring  constantly,  until  the  blue  color  is  lost  and  the  mixture 
becomes  purple,  with  a  yellow  metallic  luster  on  the  surface 
and  there  is  formed  a  finely  granular  black  precipitate. 
The  precipitate  is  the  water  insoluble  eosinates  of  methylene 
blue  and  of  methylene  azure  and  other  oxidation  products 


SOLUTIONS  AND  STAINS'  29 

of  methylene  blue.  The  end-reaction  is  reached  when  enough 
eosin  has  been  added  to  neutralize  all  of  the  methylene  blue 
and  its  oxidation  products.  To  determine  this  end-reaction, 
place  a  drop  of  the  mixture  on  a  piece  of  filter  paper,  a  slight 
eosin  halo  appears  around  the  drop,  due  to  a  slight  excess  of 
eosin.  As  the  precipitate  is  soluble  in  eosin,  add  only  enough 
excess  of  eosin  to  get  the  slight  halo.  Collect  this  precipitate 
on  a  filter  paper  and  dry  in  the  incubator  at  38°  C.  When 
thoroughly  dry,  dissolve  0.06  gram  in  20  c.c.  of  pure  methyl 
alcohol  (acetone-free).  This  is  the  stock  solution.  For 
use,  filter  the  20  c.c.  and  add  to  the  filtrate  5  c.c.  of  methyl 
alcohol. 

The  dry  powder  kteps  well:  the  alcoholic  solution  does  not 
keep  well.  Therefore  it  is  better  to  make  only  enough  of  the 
solution  to  last  a  couple  of  months. 

Method  of  Staining. — i.  Make  films  'and  dry  in  the  air. 
The  film  must  dry  quickly  and  must  be  protected  from  dust 
and  dirt. 

2.  Cover  the  dry  film  preparation  with  the  stain  for  one 
minute  (to  fix). 

3.  Add  distilled  water  to  the  stain  on  the  preparation, 
drop  by  drop,  until  a  yellow  metallic  scum  begins  to  form 
(to  stain).    Add  the  drops  of  water  rapidly  in  order  to  pre- 
vent precipitates  on  the  stained  film;  practically  add  i  drop 
of  water  for  every  drop  of  stain  used.     Allow  to  stain  for 
five  to  ten  minutes. 

4.  Wash  off  the  stain  with  distilled  water. 

5.  Wash  in  distilled  water  until  the  film  has  a  pinkish  tint. 

6.  Blot  dry  with  filter  paper.     Do  not  put  on  a  cover- 
glass  or  mount  in  liquid  petroleum. 

Red  cells  are  orange  to  pink;  nuclei,  various  shades  of  violet; 
eosinophile  granules  are  red;  neutrophile  granules  are  yellow 
to  lilac;  blood  plates  are  purplish;  malaria  parasites:  cyto- 
plasm is  blue  and  chromatin  is  metallic  red  to  rose  pink. 

Caution. — Never  heat  a  preparation  that  is  to  be  stained 


30    LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

by  Romanowsky,  and  use  only  distilled  water  or  rain  water, 
in  all  Romanowsky  methods.  Old  distilled  water  should 
be  boiled  to  drive  off  CO2,  especially  for  Giemsa  stain. 

FONTANA  METHOD  FOR  TREPONEMATA. 
The  following  three  solutions  are  used: 

I 

Acetic  acid i .  o 

Formalin 20.0 

Distilled  water 100.0 

II. 

Phenol i.o 

Acid  tannic 5.0 

III. 

Twenty-five  per  cent,  solution  of  silver 

nitrate 5.0  c.c. 

Ammonia  water i.o  drop 

Dry  slide  in  air. 

Wash  in  I  for  one  minute. 

Wash  in  water. 

Pour  on  II  and  steam  one-half  minute. 

Wash  in  water. 

Pour  on  III,  steam  for  one-half  minute. 

Wash  in  water. 

Mount  in  balsam. 

Immersion  oil  takes  out  the  color. 

CLEANSING  SOLUTION  FOR  GLASSWARE. 

Potassium  bichromate  (powdered)      .       200  grams 
Water  distilled,  up  to          ....     1500  c.c. 
Sulphuric  acid  cone 500  c.c. 

This  solution  may  be  used  for  cleaning  test-tubes  and  other 
glassware  used  in  the  preparation  of  the   Colloidal   Gold 


SOLUTIONS  AND  STAINS  31 

Reagent  and  the  Lange  test.    Microscopic  slides  cleaned  with 
this  solution  may  be  used  repeatedly. 

Method  for  preparation  of  chemically  clean  glassware: 

1.  Boil  for  thirty  minutes  in  hot  Ivory  soap  solution. 

2.  Brush  thoroughly  under  hot  tap  water. 

3.  Rinse  in  running  water  for  ten  minutes. 

4.  Immerse  in  hot  bichromate  solution  for  thirty  minutes. 

5.  Rinse  in  running  water  for  ten  minutes. 

6.  Rinse  in  ordinary  distilled  water. 

7.  Rinse  with  triply  distilled  water. 

KLOTZ'S  FLUID. 

The  chief  advantage  of  Klotz's  method  of  preserving 
pathological  specimens  is  that  the  specimens  may  be  preserved 
and  shipped  in  Fluid  No.  i. 

A  practical  method  for  a  Base  Hospital  with  a  large  patho- 
logical service  is  to  make  up  a  liberal  supply  of  Carlsbad 
salts  with  which  the  fluid  is  readily  prepared. 
Modified  Carlsbad  salts: 

Potassium  sulphate 40  grams 

Sodium  or  potassium  nitrate      .      .      .     760  grams 

Sodium  chloride 360  grams 

Sodium  bicarbonate 400  grams 

Sodium  sulphate 440  grams 

Fix  tissues  three  to  five  days  or  longer  in  Fluid  No.  i: 

Carlsbad  salts 375  grams 

Chloral  hydrate          .      .      .    -  .      .      .  375  grams 

Formalin 375  c.c. 

Water 15  liters 

Wash  three  to  five  hours  in  running  water.  Preserve  in 
Fluid  No.  2 : 


32     LABORATORY  METHODS  OF  UNITED  STATES  ARMY 

Carlsbad  salts 375  grams 

Chloral  hydrate          150  grams 

Formalin 75  c.c. 

Water 15  liters 

KAISERLING'S  FLUID. 

For  preservation  of  colors  of  gross  pathological  specimens 
the  following  method  gives  satisfactory  results: 

1.  Fix  the  tissue  in  following  solution  for  one  to  five  days 
in  Kaiserling's  Fluid  No.  i : 

Formaldehyde 200  c.c. 

Water 1000  c.c. 

Potassium  nitrate 15  grams 

Potassium  acetate         30  grams 

2.  Drain  and  place  in  80  per  cent,  alcohol  for  one  to  six 
hours. 

3.  Ninety- five  per  cent,  alcohol  for  one  to  two  hours. 

4.  Preserve  specimen  in  Kaiserling's  Fluid  No.  3: 

Potassium  acetate         .     .     .      .      .       200  grams 

Glycerine 400  c.c. 

Water 2000  c.c. 

NEUTRAL  SODIUM  HYPOCHLORITE  SOLUTION 
("DAKIN'S  SOLUTION"). 

PREPARATION  FROM  BLEACHING  POWDER. — Dakin's  Origi- 
nal Method. — A  strong  solution  of  hypochlorite  is  prepared 
by  decomposing  150  grams  bleaching  powder  (about  25  to 
35  per  cent,  available  chlorine)  with  105  grams  dry  sodium 
carbonate  (122  grams  monohydrate  (Na2CO3.H20)  or  284 
grams  washing  soda  (Na2CO3.ioH20) ).  The  mixture  is  very 
thoroughly  shaken,  both  to  make  good  contact  and  to  render 


SOLUTIONS  AND  STAINS  33 

the  precipitated  calcium  carbonate  granular  and  promote 
its  settling.  It  is  then  allowed  to  stand  quietly  and  after  half 
an  hour  the  clear  liquid  is  siphoned  off  from  the  precipitate 
and  filtered  through  paper  or  a  cotton  plug. 

A  10  c.c.  portion  is  rapidly  titrated  with  £  boric  acid  solu- 
tion (31  grams  per  liter),  using  powdered  phenolphthalein  as 
indicator  (the  usual  alcoholic  solution  of  phenolphthalein 
will  not  serve)  in  order  to  determine  the  amount  of  boric  acid 
to  be  added  to  the  rest  of  the  filtrate.  The  end-point  is  the 
disappearance  of  the  pink  color.  Each  cubic  centimeter  of 
£  boric  acid  required  for  the  10  c.c.  sample  calls  for  the 
a'ddition  of  3  grams  boric  acid  per  liter  of  filtrate.  An  excess 
of  boric  acid  should  be  avoided,  as  it  favors  the  liberation  of 
hypochlorous  acid  and  renders  the  solution  less  stable.  It  is 
best  to  add  slightly  less  than  the  calculated  amount.  The  con- 
centrated solution  thus  prepared  contains  about  4  per  cent,  of 
sodium  hypochlorite,  and  before  use  should  be  diluted  with 
about  7  parts  of  water  and  titrated  with  ^  thiosulphate  to 
determine  its  precise  hypochlorite  concentration.  It  is  then 
accurately  diluted  to  the  required  strength  (usually  0.5  to 
0.45  per  cent.). 

PREPARATION  FROM  CHLORINE  AND  SODIUM  CARBONATE. — 
Chlorine  may  be  obtained  as-  the  compressed  gas  in  steel 
cylinders  and  is  easily  measured  by  a  chlorine  meter  manu- 
factured for  the  purpose.  This  is  a  stable,  economical  and 
convenient  source  of  chlorine.  A  solution  is  prepared  con- 
taining 15  grams  of  dry  sodium  carbonate  per  liter  (=  17,6 
grams  mono  hydrate  or  40  grams  of  washing  soda.)  A 
measured  quantity,  4.8  grams  (or  about  1600  c.c.)  of  chlorine 
gas  is  allowed  to  run  into  the  solution  for  each  liter.  Ten  c.c. 
of  the  solution  is  then  titrated.  If  the  solution  is  too  weak 
more  chlorine  is  introduced.  If  the  solution  is  too  strong  it 
should  be  diluted  to  a  concentration  of  0.5  per  cent.  NaOCl 
with  1.5  per  cent,  sodium  carbonate  solution,  which  at  the 
same  time  serves  to  correct  the  unduly  diminished  alkalinity 


34     LA  BORA  TOR  Y  ME  THODS  OF  UNI  TED  S  TA  TES  ARMY 

caused  by  the  excess  of  chlorine  introduced  into  the  solution. 
If  the  final  solution  fails  to  give  a  momentary  flash  of  color 
with  alcoholic  solution  of  phenolphthalein  it  should  be 
rejected.  If  the  solution  shows  color  with  powdered  phenol- 
phthalein it  must  be  titrated  with  boric  acid  as  described 
above,  for  preparation  from  Bleaching  Powder,  until  this 
defect  is  corrected,  or  it  must  be  discarded.  The  solution 
should  be  titrated  for  hypochlorite  concentration  every 
twenty-four  to  forty-eight  hours  and  discarded  when  it  falls 
below  the  desired  lower  limit  (usually  0.45  per  cent.). 

If  a  chlorine  meter  is  not  available,  chlorine  gas  may  be  run 
into  the  1.5  per  cent,  carbonate  solution  through  any  impro- 
vised diffuser  until  the  hypochlorite  concentration  has  reached 
0.5  per  cent.  The  amount  of  chlorine  required  to  give  a  hypo- 
chlorite concentration  of  0.5  per  cent,  is  approximately  twice 
the  amount  required  to  cause  decolorization  of  powdered 
phenolphthalein.  It  is  therefore  convenient  to  add  powdered 
phenolphthalein  and  note  the  amount  of  chlorine  required  to 
cause  its  decolorization.  When  almost  twice  that  amount  of 
chlorine  has  been  introduced,  frequent  titrations  of  the  hypo- 
chlorite content  must  be  commenced  to  determine  the  proper 
point  at  which  to  stop  the  introduction  of  the  chlorine. 

TlTRATION    OF    DAKIN's     SOLUTION. — To     IO    C.C.     of    the 

Dakin  solution,  add  approximately  5  c.c.  of  a  10  per  cent, 
solution  of  potassium  iodid  and  3  c.c.  of  glacial  acetic  acid, 
lodin  is  liberated  and  dissolves  in  the  excess  of  iodid  present. 
Dilute  with  water  to  about  50  c.c.  A  standard  r^  thiosulphate 
solution  is  then  added  from  a  burette  until  the  solution  is 
just  rendered  colorless.  The  number  of  cubic  centimeters 
required  to  effect  this  result  multiplied  by  the  factor  0.0372 
gives  the  percentage  of  sodium  hypochlorite  present  in  the 
Dakin  solution. 

PREPARATION  OF  STANDARD  -^  THIOSULPHATE  SOLUTION. — 
Dissolve  exactly  24.82  grams  of  pure  crystalline  sodium  thio- 
sulphate in  water  and  make  up  to  exactly  i  liter.  One  c.c.  of 


SOLUTIONS  AND  STAINS  35 

this  standard  solution  is  equivalent  to  0.00372  gram  of 
sodium  hypochlorite. 

TITRATION  OF  BLEACHING  POWDER. — Bleaching  powders 
/ary  considerably  in  their  "available  chlorine"  content,  so 
hat  it  is  desirable  to  determine  the  available  chlorine  in  each 
arge  batch.  Bleaching  powders  with  less  than  20  per  cent, 
ivailable  chlorine  should  be  rejected.  Exceptional  samples 
nay  contain  as  high  as  35  per  cent,  available  chlorine. 

The  available  chlorine  content  may  be  determined  as  follows: 
exactly  10  grams  of  bleaching  powder  made  up  of  small 
Camples  from  different  parts  of  the  jar,  in  order  to  obtain  a 
•epresentative  sample,  are  well  shaken  with  a  liter  of  water. 
\fter  standing  about  six  hours  the  solution  is  filtered  and  a 
[o  c.c.  sample  of  the  filtrate  is  titrated  in  exactly  the  same 
nanner  as  in  the  titration  of  Dakin's  solution.  In  this  case 
he  number  of  cubic  centimeters  of  decinormal  thiosulphate 
•equired  to  decolorize,  multiplied  by  the  factor  3.55,  gives  the 
percentage  of  active  chlorine  in  the  bleaching  powder. 

OTHER  CHLORINE  ANTISEPTICS. 

Antiseptics  of  the  chlorine  group  when  properly  applied 
mve  proved  to  be  the  most  efficient  antiseptics  used  in  the 
>resent  war.  We  are  indebted  to  Dakin  for  two  other  impor- 
ant  chlorine  antiseptics: 

"  CHLORAMiN-T.1 — Chloramin-T  is  the  abbreviated  name 
ior  sodium-toluene-sulphon-chloramin.  Chloramin-T  is  sol- 
uble in  water  and  can  be  used  in  stronger  solution  (up  to  2 
)er  cent.)  than  the  hypochlorites.  It  is  more  stable  and 
txerts  more  prolonged  action  than  hypochlorite  when  acting 
n  the  presence  of  much  blood.  It  is  not  toxic  and  is  less 
rritating  than  the  hypochlorites,  but  it  has  but  little  solvent 
.ction  on  necrosed  tissue.  It  is  well  suited  for  use  on  wounds 
reviously  cleansed  with  hypochlorites,  and  in  suitably  dilute 

1  Dakin  and  Dunham:  Hand-book  of  Antiseptics. 


